Dengue virus infections are still increasing at an alarming rate in tropical and subtropical countries, underlying the need for a dengue vaccine. Although it is relatively easy to generate Ab responses to dengue virus, low avidity or low concentrations of Ab may enhance infection of FcR-bearing cells with clinical impact, posing a challenge to vaccine production. In this article, we report the characterization of a mAb, 2H12, which is cross-reactive to all four serotypes in the dengue virus group. Crystal structures of 2H12-Fab in complex with domain III of the envelope protein from three dengue serotypes have been determined. 2H12 binds to the highly conserved AB loop of domain III of the envelope protein that is poorly accessible in the mature virion. 2H12 neutralization varied between dengue serotypes and strains; in particular, dengue serotype 2 was not neutralized. Because the 2H12-binding epitope was conserved, this variation in neutralization highlights differences between dengue serotypes and suggests that significant conformational changes in the virus must take place for Ab binding. Surprisingly, 2H12 facilitated little or no enhancement of infection. These data provide a structural basis for understanding Ab neutralization and enhancement of infection, which is crucial for the development of future dengue vaccines.
Dengue is a mosquito-borne infection of the tropics and subtropics (1, 2). Some 2.5 billion people are at risk, and 50–100 million are infected annually. Most infections are either asymptomatic or result in dengue fever, a relatively mild illness. However, a much more severe form, dengue hemorrhagic fever, develops in 1–5% of infections; this can be life threatening. The incidence of dengue is increasing at an alarming rate, and epidemics can severely disrupt healthcare systems in developing countries (1, 2). Although treatment has reduced the mortality, there is still an urgent need for a vaccine.
Dengue viruses have been divided into four serotypes, differing in overall amino acid sequence by ≥30% (3, 4). Infection with one serotype does not give life-long protection against the other serotypes (5), and a hallmark of dengue infection is that dengue hemorrhagic fever is more likely to occur following a secondary infection with a heterotypic serotype, rather than following a primary infection (6). Halstead and O’Rourke (7, 8) proposed Ab-dependent enhancement (ADE) to explain this paradox, whereby an acquired humoral response to the first virus could drive a more severe clinical outcome upon a secondary exposure. There is now good evidence that cross-reactive, poorly neutralizing Abs can drive infection of FcR-bearing cells, such as monocytes, leading to increased infection and virus production (7–12).
Dengue virus has three structural proteins: capsid that encloses the positive-strand genome, and precursor membrane protein (prM) and envelope (E), both of which are components of the virion envelope structure. Abs to prM are generally poorly neutralizing but potent enhancers of infection (13, 14), whereas Abs against E show more potent neutralizing activity (15–17). E is composed of three domains (ED): I–III (18). EDI and EDII are formed by discontinuous folds at the membrane-proximal N terminus of the protein; EDII contains the fusion loop. Abs that target the highly conserved fusion loop are usually flavivirus cross-reactive (19, 20); however, because of the epitope’s inaccessibility on infectious virions, they mostly bind with low avidity and exhibit weak neutralization (20). Recently, however, a flavivirus cross-reactive mAb 2A10G6 that binds to a newly identified epitope within the fusion loop was shown to be broadly cross-neutralizing and cross-protective (21). EDIII is thought to be involved in host cell interaction (22–24), binding to heparan sulfate (25) and/or other as-yet-poorly-characterized receptor(s) (24). In mice, mAbs specific to EDIII are potent neutralizers of dengue virus (26–35), and they neutralize more strongly than do EDI- or EDII-specific Abs (33). As a result, EDIII has been considered a potential immunogen for new subunit vaccines (36–40). EDIII is a target of both serotype-specific (16, 26, 27, 32–34, 41, 42) and dengue cross-reactive (28, 30–34, 43) neutralizing Abs, although the latter tend to neutralize more weakly (28, 34).
In this article, we report a mouse mAb 2H12 that cross-reacts with the four serotypes of the dengue group and neutralizes Den1, Den3, and Den4. Crystal structures of 2H12 Fab with recombinant EDIII were determined at resolutions of 1.7, 1.8, and 3.0 Å for Den1, Den3, and Den4, respectively. They show that the Ab has a conserved mode of binding and contacts a highly conserved epitope in the AB loop of EDIII, which is largely buried in the mature virion structure, implying that gross conformational changes occur in the surface architecture of the virion upon Ab binding. This binding is temperature dependent and different across the serotypes, implying that the stability of the virus is a key factor in virus neutralization.
Materials and Methods
Expression and purification of recombinant EDIII
EDIII (aa 295–401) of dengue virus serotype 1–4, strains Hawaii, 16681, H87, and H241, were expressed in Escherichia coli and purified as described previously (44). In brief, the EDIII proteins were expressed in inclusion bodies and refolded in 100 mM Tris HCl, 500 mM l-arginine-HCl, 0.2 mM EDTA, 3.7 mM cystamine, 100 μM PMSF, and 6.6 mM 2-ME. Refolded protein was purified in PBS through a size-exclusion column (26/60 Superdex 75; GE Healthcare).
The 2H12 hybridomas were produced following standard protocols described previously (45). BALB/c female mice were immunized with 20 μg recombinant EDIII serotype 2 strain 16681 in CFA and boosted at fortnightly intervals with 20 μg protein in IFA. Three days prior to sacrifice, the mice were immunized i.v. once more. The splenocytes were fused with NS1 myeloma cells. After three rounds of single-cell cloning, mAb 2H12 was purified from the hybridoma supernatant by protein G affinity. This study was carried out in strict accordance with the recommendations in the U.K. Coordinating Committee on Cancer Research Guidelines for the Welfare of Animals. All animal procedures were approved by the Institutional Review Committee and the Home Office U.K., under the project title “Manipulation of immunity to transplanted normal and tumor grafts” (70/6874).
C6/36 cells were infected with dengue virus serotype 1 to 4 (Den1 strain Hawaii, 02-0435 and 1-0372; Den2 strain 16681, NGC and 31-178; Den3 strain H87, 01-0017 and 2-1969-9; and Den4 strain H241, 1-0093 and 1-0544). Supernatants were collected, clarified, aliquoted, and stored at −80°C.
EDIII protein or UV-inactivated virus supernatant was dotted onto nitrocellulose membrane and allowed to dry. BSA was used as an irrelevant Ag for the recombinant protein, and supernatant from mock-infected cells was used as a negative control for the virus blot. The membranes were blocked with PBS containing 0.1% Tween (PBS-T), containing an additional 5% milk for 1 h at 37°C. The membranes were then incubated overnight at 4°C with purified mAb 2H12 (5 μg/ml) in 5% milk. After washing with PBS-T, the membranes were incubated with anti-mouse IgG-HRP–conjugated Ab (Sigma-Aldrich) for 1 h at 37°C. Dots were visualized using ECL Plus (PerkinElmer).
Binding affinity of 2H12 Ab
The Kd of mAb 2H12 against EDIII was calculated as described previously (44). In brief, recombinant EDIII protein was coated onto plates, and a direct ELISA with various concentrations of 2H12 was performed. BSA was used as an irrelevant Ag, and the values observed against this were subtracted from the values detected against the EDIII. For each Ag, the percentage of maximum OD was calculated, and the dissociation constants were determined (Prism Software). The virus-specific Kd values were determined by capture ELISA. Plates were coated with cross-reactive human anti-dengue mAb 751.B3 (generated in our laboratory) overnight at 4°C. Plates were rinsed three times with PBS-T and blocked for 1 h at 37°C with 200 μl blocking buffer (3% BSA in PBS). Plates were incubated for 1 h at 37°C with supernatant from virus- or mock-infected C6/36 cells diluted in dilution buffer (0.5% BSA in PBS-T), washed, and incubated with 50 μl 2-fold serial dilutions of mAb 2H12 for 1 h at 37°C. The plates were then incubated for 1 h at 37°C in 50 μl anti-mouse IgG-alkaline phosphatase-conjugated secondary Ab (Sigma-Aldrich), followed by p-nitrophenyl phosphate substrate (SIGMA FAST; Sigma-Aldrich). The reaction was quenched, and the virus was inactivated by incubation with 0.4 M NaOH for 20 min at room temperature. Background mock readings were subtracted. Where a plateau was observed, the percentage of maximum OD was calculated, and the dissociation constants were determined as the concentration of Ab required for 50% binding (Prism Software).
Focus reduction neutralization test
Micro-focus reduction neutralization tests (FRNTs) were conducted on Vero cells, as described previously (46). The FRNT50 titer was defined as the concentration of Ab that reduced the number of foci by 50%.
Supernatants from dengue- or mock-infected C6/36 cells were captured onto plates coated with 2H12 and incubated for 1 h at 4, 30, or 37°C. Plates were then incubated with pooled convalescent dengue hyperimmune serum (hemagglutination titer ≥ 1/25,600) to detect bound virus, followed by alkaline phosphatase-conjugated anti-human IgG. The reaction was developed by the addition of p-nitrophenyl phosphate substrate and quenched with 0.4 M NaOH for 20 min at room temperature. The absorbance was read at 405 nm.
Ab-dependent enhancement assay
Ten-fold serial dilutions of mAb 2H12 were incubated with an equal volume of virus for 1 h at 37°C, at a multiplicity of infection of 0.2. The anti-envelope mAb 4G2 was used as a positive control. The Ab:virus mixture was transferred onto U937 cells in a 24-well plate and incubated at 37°C for 4 d (47). Supernatants were harvested, and their viral titer was assessed by a focus-forming assay on Vero cells. The infected culture supernatants were serially diluted and incubated with Vero cells in a 96-well plate for 2 h at 37°C. The monolayers were then overlaid with 1.5% carboxymethylcellulose, in Eagle’s MEM tissue culture medium, and incubated at 37°C for 3 d. Virus foci were stained with mAb 4G2, followed by peroxidase-conjugated anti-mouse Ig, and they were visualized by the addition of diaminobenzidine substrate. The infecting fold enhancement is calculated by dividing focus forming unit (ffu)/ml in the presence of Ab by ffu/ml in the absence of Ab.
The sequences of the 2H12 H chain and L chain variable regions were determined using 5′-RACE (Invitrogen) (48). In brief, the mRNA was extracted from the 2H12 hybridoma cells, and cDNA was created using a poly T primer. Gene-specific primers, complementary to the CH1 region of each chain, were then used as 3′ primers to amplify the variable regions from the cDNA.
Crystallographic structure determination
Purified 2H12 Fab, prepared by papain digestion of mAb, was incubated with EDIII for 1 h at room temperature, followed by purification by size-exclusion chromatography. The protein complexes were concentrated and set up in crystallization experiments, using the sitting vapor-diffusion method (49). The complexes were mixed with 10% glycerol prior to crystallization to slow nucleation and improve crystal size, because initial crystallization trials resulted in microcrystals. All crystals used for data collection grew at 21°C with 20% w/v PEG3350 used as precipitant and various salts (0.2 M potassium thiocyanate for 2H12–EDIIID1 and 2H12–EDIIID4, 0.2 M di-hydrogen phosphate for 2H12–EDIIID3). Protein crystals were cryoprotected with 20% glycerol. X-ray data were collected at 100 K at Diamond Light Source (Beamline I04), as a series of 1° oscillations. Diffraction data were processed using either HKL2000 (50) or xia2 (51). The structures of the complexes were solved by molecular replacement using Phaser (52). For each Fab–Ag complex, the structure was determined using three search models: one for the C region of Fab (CH1 and CL), one for the V region of Fab (VH and VL), and one for EDIII Ag. The Fab was divided into two search models because of flexibility of the elbow between variable and constant domains (53). The complex of 2H12–EDIIID3 was solved using component coordinates (pdb code 1A3R and 1UZG [residues 296–394] for the Fab and EDIIID3, respectively). The refined structure of 2H12–EDIIID3 was used as a search model for molecular replacement of 2H12–EDIIID1 and 2H12–EDIIID4. The complex structure was again separated into three search models comprising the Fab constant domains, the Fab variable domains, and EDIII.
Atomic positions and their associated B factors were refined using Refmac 5 (54–56) and Buster (57) and were rebuilt in Coot (58). Noncrystallographic local structure similarity restraints were used during the refinement of all structures. The quality of the models was analyzed with MolProbity (59), and the binding surfaces were analyzed with PISA (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html) (60) (Supplemental Table III).
Characterization of mAb 2H12
mAb 2H12 was produced from BALB/c mice immunized with recombinant EDIII from dengue serotype 2 (EDIIID2) strain 16681. 2H12, an IgG2b Ab, was found to be cross-reactive against the four dengue virus serotypes by dot blot, recognizing both the recombinant EDIII Ag (Fig. 1A) and the whole virus (Fig. 1B).
The neutralizing effects of 2H12 were tested on three strains of each dengue serotype. Neutralization was observed against all three strains of serotypes 1 and 4, as well as against a single strain 01-0017 of Den3 (Fig. 2A–D), but not against the three strains of serotype 2. The mean (n = 2–3) concentration of Ab required for 50% neutralization was between 0.56 and 54 nM for Den1, 29 nM for Den3 strain 01-0017, and 145 nM for Den4 strain H241. This was somewhat weaker than the 4G2 positive control (Fig. 2E) and weaker than a Den2-specific anti-EDIII mouse mAb 3H5 (1.2–4.6 nM) (28, 34).
In addition, Ab-dependent enhancement assays were performed on U937 cells, a human monocytic cell line expressing FcγRII (61, 62), which binds mouse IgG2b Abs (61). In the absence of Ab, these cells show a very low level of infection (0.01–1% infected cells or 10–100 ffu of progeny/ml at a multiplicity of infection of 1). To our surprise, the presence of 2H12 resulted in little or no enhancement of infection in U937 cells (Fig. 2F). In comparison, the control mAb 4G2 exhibited enhancements of between 50- and 2500-fold (Fig. 2G).
The avidity of full-length 2H12 for EDIII was determined by ELISA with immobilized recombinant EDIII protein (Fig. 1C). The binding avidity of 2H12 for EDIII of the four serotypes was very similar, with Kd values of 0.45, 0.42, 0.47, and 0.42 nM for EDIIID1, D2, D3, and D4, respectively.
Finally, ELISA assays were performed on whole-virus particles for all four serotypes, using 2H12 Ab (Fig. 1D). Unlike for the recombinant EDIII, there were differences in the measured Kd values. The strongest binding was against serotype 1 (1.90 nM), followed by serotype 4 (13.81 nM). The binding against serotypes 2 and 3 did not reach saturation, and so the Kd were estimated at >125 and >62.5 nM, respectively. The similar binding affinities of 2H12 for EDIII, as well as the differences in binding for whole-virus particles, suggests that the differences in neutralization are due to differences in some property of the virions.
The structures of 2H12–EDIII complexes
Fab fragments of 2H12 were mixed with EDIII from the four serotypes and used in crystallization trials. Dengue strains Hawaii, 16681, H87, and H241 were used for serotypes 1, 2, 3, and 4, respectively. Three of four 2H12–EDIII complexes yielded crystal structures: EDIIID1, D3 and D4. Unfortunately, all diffracting crystals of the putative 2H12–EDIIID2 complex were composed of Fab alone, despite 2H12 forming a stable complex prior to crystallization (data not shown).
The crystals of 2H12–EDIIID1 and 2H12–EDIIID3 diffracted to 1.7 and 1.8 Å, respectively (Fig. 3A, Table I), and each contained one copy of the Fab–EDIII complex. The crystals of 2H12–EDIIID4 diffracted to a lower resolution (3.0 Å) and contained four copies of the Fab–EDIII complex. The structures of the complexes were solved by molecular replacement and refined with residuals R = 17.1, Rfree= 19.8, R = 18.4, Rfree= 21.7; and R = 19.6, Rfree= 25.2 for serotypes 1, 3, and 4, respectively (Table I). The stereochemistry of the refined models was excellent, with residues in favored region/outliers of the Ramachandran plot: 98.3%/0.00% for 2H12–EDIIID1, 97.9%/0.21% for 2H12–EDIIID3, and 95.4%/1.00% for 2H12–EDIIID4.
Binding of the Fab did not significantly change the overall structure of EDIII (Fig. 3), with root mean square deviation (r.m.s.d.) of Cα of 1.23, 0.92, and 1.58 Å for EDIIID1, EDIIID3, and EDIIID4, respectively, compared with the published atomic models (pdb codes 3IRC – EDIIID1, 1UZG – EDIIID3, and 2H0P – EDIIID4). The main conformational change occurred in the AB loop, with Gln316 shifted by 2.98, 3.00, and 3.44 Å in EDIIID1, EDIIID3, and EDIIID4, respectively. Moreover, the structures of the three EDIII proteins are very similar compared with each other, with r.m.s.d. of Cα of 0.84–1.18 Å (Supplemental Table I), despite 34–43% difference between the amino acid sequences.
However, the elbow angle of the bound Fab (the angle between constant and variable domains) differs between the three complexes and is 172, 155, and 144° for 2H12–EDIIID1, 2H12–EDIIID3, and 2H12–EDIIID4, respectively (Supplemental Fig. 1A), perhaps reflecting the different crystal packing of the three complexes. Despite the differences in the elbow angle, the variable (VHVL) and constant (CH1CL) regions of the Fab structures are very similar in the three complexes (Supplemental Table I).
The 2H12 epitope
Two structures of Fabs complexed to EDIII of flaviviruses have been solved. E16 is a murine mAb that binds to the so-called “lateral ridge epitope” of West Nile virus (WNV) EDIII, defined by four discontinuous regions, including the N-terminal linker region (residues 302–309) and three strand-connecting loops: BC (residues 330–333), DE (residues 365–368), and FG (residues 389–391) (Fig. 4A) (63, 64). It is thought to be prototypic for a number of high-avidity and potently neutralizing Abs to both dengue virus and WNV. Ab 1A1D-2, specific for dengue serotypes 1–3, binds to an epitope on the A strand of EDIII (30, 34). The 2H12 epitope footprints on the EDIII proteins are presented as a gradient of buried surface area of residues in Fig. 3B. Between these three serotypes, the mode of engagement of 2H12 Fab is very similar, with only minor differences in the contact surface area. For the 2H12–EDIIID1, 2H12–EDIIID3, and 2H12–EDIIID4 complexes, the surface area of interaction of EDIII was 549, 567, and 521 Å2, respectively, and the surface area of interaction of 2H12 was 652, 448, and 394 Å2. This interface area is significantly smaller than that observed for 1A1D-2 (30) and E16 (64) (905 and 782 Å2, respectively).
EDIII binds to 2H12 Fab predominantly via the AB loop (residues 314–317), which is inserted into a groove formed between the H chain and L chain of Fab. Residues from strands A and E and the D-Dx loop are also involved in contacts with the CDR of 2H12 (Fig. 4A). Ab–Ag binding is mediated by hydrogen bonds and van der Waals interactions formed between seven fully conserved residues (Fig. 4A, Supplemental Table II), Lys310, Glu314, Thr315, Gln316, His317, Ile352, and Glu368, and four CDRs, three from the H chain (residues 33–35, 57–59, and 100–101) and one from the L chain (residues 94–96). An example of the interface between EDIIID1 (Gln316/His317) and 2H12 is presented in Supplemental Fig. 1B. In addition, the structures of 2H12–EDIIID1 and 2H12–EDIIID3 determined at higher resolutions also indicate possible water-mediated hydrogen bonds engaging Val312, Gln/Lys323, and Asn366 and van der Waals interactions mediated by Ala/Ser313, Val/Leu321, and Ala/Ser354 (Supplemental Table II). However, the relatively large distances between these residues (>3.5 Å) suggests that their contribution to the energy of binding may be limited; furthermore, because the avidity of 2H12 to EDIII is very similar across all four serotypes (Fig. 1C), differences in these water-mediated interactions is unlikely to differentially modulate the strength of the interaction.
Although 2H12 has an epitope distinct from 1A1D-2 (Fig. 4), their epitopes overlap by one residue: Lys310 (30, 34). The residue has been shared by several anti-EDIII mAbs that recognize the strand A, such as 1A1D-2, 9D12, and 4E11 (17, 28, 32, 34, 43, 65). This residue forms a hydrogen bond, via the ε-NH3(+) group, with the carbonyl oxygen of Val58 of 2H12 H chain and the carbonyl oxygen of Lys30 or Asp52 of 1A1D-2 H chain (30).
When modeled onto the structure of mature dengue virions, the epitope footprint of 2H12 revealed the likely basis for its poor neutralization. The epitope is not accessible on the surface of the virus but forms the dimer interface between EDI and EDIII (Fig. 5A, 5B, Supplemental Table III). Only a small area of the epitope is exposed on the model of the prefusion mature virion (Fig. 5C, 5D). Furthermore, the residues involved in the core AB loop epitope, which are buried in the mature virus, are not solvent accessible on the trimeric prM-E spikes of the immature virus (pdb code 3C5X, 3C6D) or the trimeric E spikes in the postfusion conformation (pdb code 1OK8, 3G7T) (Supplemental Table III).
The effect of temperature on binding
The relatively “inaccessible” epitope for mAb 1A1D-2 can be exposed by a change in virus conformation or “virion breathing,” which was shown to be temperature sensitive (30). Therefore, we tested the effect of temperature on 2H12 binding (Fig. 2H). For all serotypes, a reduction in temperature resulted in a reduction in binding, with ∼30% lower binding to Den1, Den3, and Den4 at 4°C. Interestingly, the binding to Den2 was the most temperature sensitive, with a 73% reduction at 4°C (Fig. 2H). Of note, 2H12 binding to Den1 was ∼2-fold higher than to the other serotypes.
Much of the current interest in dengue virus is driven by the underlying need for a vaccine or effective treatment. Although antivirals have a role to play, the development of effective vaccines is key to the prevention and spread of this important human pathogen. Having a structural basis for understanding Ab neutralization, as well as correlating these effects with Ab-dependent enhancement, will be crucial in the development of dengue vaccines.
In this article, we functionally characterized a mAb, 2H12, raised in mice against EDIII of dengue virus serotype 2. 2H12 can bind to the four serotypes. The neutralizing potential of 2H12 is lower than a number of other DIII Abs (27, 28, 34), but, in contrast, it showed no Ab-dependent enhancement activity. Alongside these functional data, we reported the structures of the 2H12 Fab complexed with EDIII, revealing the core epitope underlying recognition by this cross-reactive anti-dengue mAb. This epitope is largely buried in the interface between EDIII and EDI of the prefusion mature virus (as determined by cryo-electron microscopy), with only 18% of the epitope surface exposed to solvent (Fig. 5). All seven of the contact residues for 2H12 are conserved between the four virus serotypes, despite the significant sequence and functional differences between serotypes. The epitope centers on a core ETQH motif within the conserved six-amino acid sequence 314ETQHGT319, in the AB loop of dengue EDIII. This region was suggested to contain binding epitopes and was detected through yeast-display mapping of a panel of EDIII Abs (34). Although these Abs were cross-reactive, and, in some cases also targeted WNV, they were generally poor neutralizers, consistent with our findings for 2H12.
In addition, Lys310, which forms direct hydrogen bonds with Val58 of the H chain and forms part of the strictly conserved epitope, may significantly contribute to neutralization by 2H12. Interestingly, Lys310 also contributes to the epitopes recognized by other known cross-reactive anti-EDIII Abs that recognize strand A of EDIII, such as 1A1D-2, 4E11, 9D12, and WN E114, suggesting that Lys310 is an important residue for this Ab group (30, 34, 66).
The neutralizing potency of 2H12 is low compared with other mAb binding to EDIII, particularly those binding to the lateral ridge, such as E16 that targets WNV (64). Furthermore, despite having a conserved epitope, 2H12 neutralized Den1 and 4 and one strain of Den3, but it showed no neutralization of Den2. Similarly, recent studies showed that neutralizing capacity varies between virus strains of the same serotype (26, 32, 33, 35), despite the epitopes being well conserved and, in some cases, the binding being similar (32). The difference in 2H12 virus binding and neutralization contrasts with the very similar binding affinity of 2H12 to EDIII, despite 28–46% differences between serotypes for this domain, and it must reflect differences in the structural stability and dynamics of the dengue virions (Fig. 5).
The fact that 2H12 can bind to the virion implies that there must be conditions under which the buried AB loop epitope becomes accessible to the Ab. It is established that icosahedral viruses, such as picornaviruses and nodaviruses, are dynamic particles and can undergo “breathing” motions (away from low-energy structures determined by crystallography) (67, 68). Further insight into conformational breathing in flaviviruses has come from structures of Fabs E16 and 1A1D-2 complexed to WNV and dengue virus, respectively. The structure of E16 Fab bound to WNV revealed that its lateral ridge is fully exposed (in 120 of the 180 copies in the virus) and needs little or no conformational change in the intact virion for binding (63, 64). In contrast, 18% of the binding surface of 1A1D-2 is obscured in the mature virion structure, and full saturation of dengue virus by 1A1D-2 Fab only occurred when increasing the temperature to 37°C (30). A cryo-electron microscopy structure of this complex revealed that binding of 120 copies of 1A1D-2 Fab resulted in all 180 copies of E undergoing major conformational rearrangements, with complete distortion of the virus compared with its native prefusion form. This conformational change is temperature sensitive, and it was proposed that increased “breathing” of the virus capsid would allow the exposure of buried epitopes (30).
The binding of 2H12 was also shown to be temperature sensitive. This temperature dependence differed between serotypes, much like neutralizing ability, with significantly poorer binding to Den2 occurring at lower temperatures. This temperature-dependent difference in Ab binding between Den2 and the other serotypes is intriguing and may reflect a more rigid Den2 capsid, and it possibly explains the inability of 2H12 to neutralize this serotype.
A model in which the virus “breathes” between the prefusion mature structure and various antigenically altered forms, when Ab binds, may also provide an explanation for the lack of ADE observed for 2H12. ADE occurs by uptake of Ab-bound virus by FcR-bearing cells. If the structural distortions that are locked in place by Fab binding are sufficient to prevent the rearrangements required for fusion, ADE will not occur. Alternatively, at low Ab concentrations (the point at which ADE usually occurs), Ab occupancy may not be sufficient to facilitate ADE. Recently, it was shown that TRIM21 interacts with the Fc part of Ab bound to adenovirus, leading to virus degradation inside cells. This suggests that TRIM21 may play a role in neutralization and ADE of dengue virus (69).
Recent studies suggest that the EDIII-specific response in humans following infection actually plays less of a role than first thought (44, 70–72). Although the EDIII-specific Ab response can be easily measured in human serum, depletion of these Abs does not reduce the neutralizing or enhancing capacity of the serum (44, 72). Despite this, EDIII remains an attractive vaccine target, because Abs that target certain regions are often of high avidity and can be potently neutralizing. When sequential immunizations are carried out in mice with recombinant EDIII alone, the neutralizing response is considerably weaker than that induced against the intact virion (73). However, when EDIII is used to boost responses following priming with the intact virion, neutralizing responses are significantly higher than when using the intact virion alone (73). Therefore, EDIII will likely make an excellent boosting Ag by strengthening the high-avidity and functionally relevant response that is primed by the virion.
2H12 was generated by immunization with recombinant EDIII. Although its epitope is not fully exposed on the intact virion, 2H12 is likely to be typical of some Abs induced by an EDIII subunit vaccine. An epitope such as this, although weakly neutralizing, may well be beneficial in vaccines, because it does not facilitate ADE. It is hoped that structural studies, such as ours, may further our understanding of cross-neutralization, as well as the structural basis for ADE, critical for the design of effective vaccines in the future.
The authors have no financial conflicts of interest.
We thank W. Lu for help with tissue culture, K. Harlos and T. Walter for crystallization expertise, the staff at Diamond Light Source beamline I04 for support in data collection, and Dave Stuart for useful discussions and critical reading of the manuscript. We also thank P. Malasit and S. Noisakran for providing DENV strains 02-0435, 1-0372, 01-0017, 2-1969-9, 1-0093, and 1-0544 and C. Simmons for strain 31-178.
This work was supported by the Medical Research Council, the Wellcome Trust, the National Institute for Health Research Biomedical Research Centre Funding Scheme, and the Office of the Higher Education Commission and Mahidol University under the National Research Universities Initiative. The Wellcome Trust is also acknowledged for providing administrative support (Grant 075491/Z/04).
The coordinates presented in this article have been submitted to the Protein Data Bank (http://www.rcsb.org/pdb/home/home.do) under accession numbers 4al8, 4ala, and 4am0. The EDIII sequences of Den1 strains 02-0435 and 1-0372, Den2 strain 31-178, Den3 strains 01-0017 and 2-1969-9, and Den4 strains 1-0093 and 1-0544 used in this study were submitted to GenBank (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers JQ740878, JQ740879, EU482654.1, JQ740880, JQ74088, JQ740882, and JQ740883.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- Ab-dependent enhancement
- envelope domain
- focus-forming unit
- focus reduction neutralization test
- PBS containing 1% Tween
- precursor membrane protein
- root mean square deviation
- West Nile virus.
- Received January 19, 2012.
- Accepted March 19, 2012.
- Copyright © 2012 by The American Association of Immunologists, Inc.